Innate Ligands Research Articles

Molecular Docking of Brazilin from Secang Wood Plant (Caesalpinia sappan L.) as an Anti-Breast Cancer

Breast cancer is a disease characterized by abnormal cell growth, which can invade adjacent tissues or metastasize to other organs. Secang (Caesalpinia sappan L.) is a plant that has been used as an alternative medicine for a variety of health conditions, including some types of cancer. This study aims to determine whether the brazilin compound found in secang wood can interact with the target receptors estrogen alpha, 17-β-HSD-1, and NUDT5, potentially serving as an anticancer candidate. Molecular docking simulations were employed to identify the molecular interactions of brazilin against estrogen receptor alpha (ERα) (PDB ID: 3ERT), 17β-HSD-1 (PDB ID: 3HB5), and NUDT5 (PDB ID: 5NQR) using AutoDockTools software. The results showed that the best free-binding energy (ΔG) value obtained for brazilin against the 17-β-HSD-1 receptor was -9.16 kcal/mol, with an inhibition constant of 192.45 nM. The ΔG value of brazilin with estrogen alpha was -6.68 kcal/mol, and the ΔG value for brazilin against NUDT5 was -4.8 kcal/mol. Brazilin has a higher potency compared to innate ligands based on the docking result of estrogen alpha receptor and NUDT5. Some structural similarities and interactions occurred between the amino acids GLY186 and TYR155 in brazilin with the binding of amino acids formed in the innate ligand against the 17-β-HSD-1 receptor, thus showing similar affinity to the 17-β-HSD1 receptor. In silico approaches provided valuable insights into the potential of brazilin as an anti-breast cancer agent.

Synergistic Targeting of Innate Receptors TLR7 and NOD2 for Therapeutic Intervention in Multiple Sclerosis.

Regulation of neuroinflammation is critical for maintaining central nervous system (CNS) homeostasis and holds therapeutic promise in autoimmune diseases such as multiple sclerosis (MS). Previous studies have highlighted the significance of selective innate signaling in triggering anti-inflammatory mechanisms, which play a protective role in an MS-like disease, experimental autoimmune encephalomyelitis (EAE). However, the individual intra-CNS administration of specific innate receptor ligands or agonists, such as for toll-like receptor 7 (TLR7) and nucleotide-binding oligomerization-domain-containing protein 2 (NOD2), failed to elicit the desired anti-inflammatory response in EAE. In this study, we investigated the potential synergistic effect of targeting both TLR7 and NOD2 simultaneously to prevent EAE progression. Our findings demonstrate that simultaneous intrathecal administration of NOD2- and TLR7-agonists led to synergistic induction of Type I IFN (IFN I) and effectively suppressed EAE in an IFN I-dependent manner. Suppression of EAE was correlated with a significant decrease in the infiltration of monocytes, granulocytes, and natural killer cells, reduced demyelination, and downregulation of IL-1β, CCL2, and IFNγ gene expression in the spinal cord. These results underscore the therapeutic promise of concurrently targeting the TLR7 and NOD2 pathways in alleviating neuroinflammation associated with MS, paving the way for novel and more efficacious treatment strategies.

Abstract 5228: Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile

Abstract Acute myeloid leukemia (AML) is an aggressive hematological malignancy that, despite advances in treatment, has an overall 5-year survival rate of less than 30%. There is an urgent unmet need for new and effective therapies for AML. Chimeric antigen receptor (CAR)-modified cell therapies are emerging as promising options. However, the selection of suitable antigens is challenging, due to the heterogeneous expression of potential antigen targets in AML. Most antigens, such as CD123, are expressed on both leukemic cells and normal hematopoietic stem and progenitor cells. In addition, conventional autologous or allogeneic T cell therapies present many limitations, such as cytokine release syndrome (CRS) and graft-vs-host disease (GvHD). We therefore evaluated CD123-targeting CAR designs in vδ1-enriched γδT cells, an innate immune cell platform that is inherently non-MHC restricted, has low risks of CRS and GvHD, and is capable of discriminating between malignant and healthy cells in preclinical models. We engineered vδ1-enriched γδT cells from peripheral blood of healthy individuals to express various CD123-targeting CAR constructs. The CD123-targeting CAR-engineered vδ1-enriched γδT cells expressed high levels of natural cytotoxicity receptors, suggesting capability to target innate receptor ligands on AML cell surface. CD123-targeting CAR-expressing vδ1-enriched γδT cells demonstrated enhanced cytotoxicity in vitro against multiple AML cell lines, in both short-term co-culture and repeated antigen stimulation assays. CD123-targeting CAR-expressing vδ1-enriched γδT cells also showed enhanced efficacy and survival in vivo against mouse models of human AML. Interestingly, attenuation of CAR activity via novel designs preserved the inherent selectivity of vδ1-enriched γδT cells against AML cells relative to normal hematopoietic stem or progenitor cells, which also express CD123. Together, our results demonstrated through rational CAR designing and engineering in vδ1-enriched γδT cells, an allogeneic cell therapy targeting CD123 can be generated with selective cytotoxicity against AML cells while sparing normal hematopoietic stem cells. Such product designs have the potential to be a transformative therapeutic option for a broad population of AML patients. Citation Format: Mei Rosa Ng, Sarah L. Hayward, Di Zhang, Gary Gu, Bri Paulhus, Donjeta Gjuka, Collin Crowley, Sophie McLaughlin, Anya Lublinsky, Angel Mathew, Evans Berreondo Giron, Doanh Mai, Madisen Lee, Megha Wal, Rebecca Alade, Andre Simoes, Robin Lytle, James Gavin, Shira Cramer, Zhong-hua Yan, Taylor Hickman, Rashmi Choudhary, Cori Abikoff, Yana Wang, Istvan Kovacs, Nandhu M. Sobhana, Lan Cao. Engineering CD123-targeting vδ1-enriched γδT cells with enhanced preclinical efficacy and safety profile [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5228.

Spatiotemporal Patterns of Substance P-Bound MRGPRX2 Reveal a Novel Connection Between Macropinosome Resolution and Secretory Granule Regeneration in Mast Cells.

MRGPRX2, the human member of the MAS-related G protein coupled receptors (Mrgprs), serves as the cellular target of human mast cells (MCs) for innate ligands, including neuropeptides and antimicrobial peptides. In addition, MRGPRX2 also functions as the receptor for multiple FDA-approved drugs. As such, MRGPRX2 is a mediator of MC responses in neurogenic inflammation, host defense and pseudoallergy. We analyzed the spatiotemporal patterns of MRGPRX2 following its binding of the neuropeptide substance P (SP). Herein, we show that MRGPRX2 internalizes via both endocytosis and macropinocytosis, followed by its distribution between a perinuclear region and the secretory granules (SGs). Further, we show that MRGPRX2-containing macropinosomes undergo resolution by a mechanism that involves dynamin and LC3, giving rise to the incorporation of both LC3 and MRGPRX2 into the SGs. SP then promotes the acidification of the LC3-associated SGs, presumably by stimulating their fusion with lysosomes. Taken together, our results reveal a unique mode of MRGPRX2 trafficking that complements endocytosis and involves macropinocytosis, autophagic machinery-assisted macropinosome resolution and receptor delivery to the SGs.

Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis.

The term 'uveitis' describes a heterogeneous set of conditions that all feature intraocular inflammation. Broadly, uveitis is defined by etiology: infection or autoimmunity. Infectious uveitis requires treatment with the appropriate antimicrobial agents, while autoimmune uveitis requires treatment with corticosteroids or other immunosuppressive agents. Post-infectious uveitis is a form of chronic uveitis that requires corticosteroids to control immune sequela following the initial infection. Uveitis associated with Mycobacterium tuberculosis (Mtb) infection is a well-recognized form of post-infectious uveitis, but the mechanisms of disease are not fully understood. To understand the role mycobacterial antigens and innate ligands play in stimulating chronic ocular inflammation following mTB infection, the model Primed Mycobacterial Uveitis (PMU) was developed for use in mice. This manuscript outlines the methods for generating PMU and monitoring the clinical course of inflammation using color fundus and optical coherence tomography (OCT) imaging. PMU is induced by immunization with heat-killed mycobacterial extract followed by intravitreal injection of the same extract into one eye seven days later. Ocular inflammation is monitored longitudinally using in vivo imaging and followed by sample collection for a wide range of assays, including histology, flow cytometry, cytokine analysis, qPCR, or mRNA sequencing. The mouse model of PMU is a useful new tool for studying the ocular responses to mTB, the mechanism of chronic uveitis, and for preclinical effectiveness tests of new anti-inflammatory therapies.

Primed Mycobacterial Uveitis (PMU) as a Model for Post-Infectious Uveitis

The term 'uveitis' describes a heterogeneous set of conditions that all feature intraocular inflammation. Broadly, uveitis is defined by etiology: infection or autoimmunity. Infectious uveitis requires treatment with the appropriate antimicrobial agents, while autoimmune uveitis requires treatment with corticosteroids or other immunosuppressive agents. Post-infectious uveitis is a form of chronic uveitis that requires corticosteroids to control immune sequela following the initial infection. Uveitis associated with Mycobacterium tuberculosis (Mtb) infection is a well-recognized form of post-infectious uveitis, but the mechanisms of disease are not fully understood. To understand the role mycobacterial antigens and innate ligands play in stimulating chronic ocular inflammation following mTB infection, the model Primed Mycobacterial Uveitis (PMU) was developed for use in mice. This manuscript outlines the methods for generating PMU and monitoring the clinical course of inflammation using color fundus and optical coherence tomography (OCT) imaging. PMU is induced by immunization with heat-killed mycobacterial extract followed by intravitreal injection of the same extract into one eye seven days later. Ocular inflammation is monitored longitudinally using in vivo imaging and followed by sample collection for a wide range of assays, including histology, flow cytometry, cytokine analysis, qPCR, or mRNA sequencing. The mouse model of PMU is a useful new tool for studying the ocular responses to mTB, the mechanism of chronic uveitis, and for preclinical effectiveness tests of new anti-inflammatory therapies.

Abstract SY20-01: USP18 modulates the ISGylome immune signaling, and sensitizes tumor cells to irradiation and CTL recognition

Abstract Interferon (IFN) signalling pathways, a key element of the innate immune response, contribute to resistance to conventional chemotherapy, radiotherapy, and immunotherapy, and are often deregulated in cancer. The deubiquitylating enzyme USP18 is a major negative regulator of the IFN signalling cascade and is the predominant human protease that cleaves ISG15, a ubiquitin-like protein tightly regulated in the context of innate immunity, from its modified substrate proteins in vivo. In this study, using advanced proteomic techniques, we have significantly expanded the USP18-dependent ISGylome and proteome in a chronic myeloid leukaemia (CML)-derived cell line. USP18-dependent effects were explored further in CML and colorectal carcinoma cellular models. Novel ISGylation targets were characterised that modulate the sensing of innate ligands, antigen presentation and secretion of cytokines. Consequently, CML USP18-deficient cells are more antigenic, driving increased activation of cytotoxic T lymphocytes (CTLs) and are more susceptible to irradiation. Our results provide strong evidence for USP18 in regulating antigenicity and radiosensitivity, highlighting its potential as a cancer target. Citation Format: Benedikt Kessler. USP18 modulates the ISGylome immune signaling, and sensitizes tumor cells to irradiation and CTL recognition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr SY20-01.

Innate Signaling in the CNS Prevents Demyelination in a Focal EAE Model.

The pathological hallmark of multiple sclerosis (MS) is the formation of multifocal demyelinating lesions in the central nervous system (CNS). Stimulation of innate receptors has been shown to suppress experimental autoimmune encephalomyelitis (EAE), an MS-like disease in mice. Specifically, targeting Toll-like receptor 9 (TLR9) and NOD-like receptor 2 (NOD2) significantly reduced disease severity. In the present work we have developed a novel focal EAE model to further study the effect of innate signaling on demyelinating pathology. Focal lesions were induced by stereotactic needle insertion into the corpus callosum (CC) of mice previously immunized for EAE. This resulted in focal pathology characterized by infiltration and demyelination in the CC. We find that intrathecal delivery of MIS416, a TLR9 and NOD2 bispecific innate ligand, into the cerebrospinal fluid reduced focal lesions in the CC. This was associated with upregulation of type I and II interferons, interleukin-10, arginase-1, CCL-2 and CXCL-10. Analysis of draining cervical lymph nodes showed upregulation of type II interferons and interleukin 10. Moreover, intrathecal MIS416 altered the composition of early CNS infiltrates, increasing proportions of myeloid and NK cells and reducing T cells at the lesion site. This study contributes to an increased understanding of how innate immune responses can play a protective role, which in turn may lead to additional therapeutic strategies for the prevention and treatment of demyelinating pathologies.

Innate IL-23/Type 17 immune responses mediate the effect of the 17q21 locus on childhood asthma.

Several childhood asthma risk loci that relate to immune function have been identified by genome-wide association studies (GWAS), but the underlying mechanisms remain unknown. Here, we examined whether perturbed innate immune responses mediate the association between known genetic risk variants and development of childhood asthma. Peripheral blood mononuclear cells from 336six-month-old infants from the Copenhagen Prospective Studies on Asthma in Childhood (COPSAC2000 ) cohort were stimulated in vitro with six different innate ligands (LPS, CpG, poly(I:C), R848, HDMAPP and aluminium hydroxide together with low levels of LPS) followed by quantification of 18 released cytokines and chemokines 40h after the stimulations. The innate immune response profiles were decomposed by principal component (PC) analysis, and PC1-5 were used in mediation analyses of the effect of 25 known genetic risk variants on childhood asthma until age 7. The effects of two variants from the 17q21 locus (rs7216389, rs2305480) on asthma and exacerbation risk were significantly mediated by immune parameters induced in response to ligands mimicking intracellular colonization; bacterial DNA (CpG) and double-stranded viral RNA (poly(I:C)). The Th17 and innate lymphoid cell type 3-amplifying cytokine IL-23 was the most prominent cytokine involved. The 17q21 effect on childhood asthma and exacerbations was partly mediated by deregulation of IL-23 in response to intracellular microbial ligands, which may suggest ineffective clearance of intracellular pathogens in the lungs.

The microbiota plays a critical role in the reactivity of lung immune components to innate ligands.

The gut microbiota contributes to shaping efficient and safe immune defenses in the gut. However, little is known about the role of the gut and/or lung microbiota in the education of pulmonary innate immune responses. Here, we tested whether the endogenous microbiota in general can modulate the reactivity of pulmonary tissue to pathogen stimuli by comparing the response of specific-pathogen-free (SPF) and germ-free (GF) mice. Thus, we observed earlier and greater inflammation in the pulmonary compartment of GF mice than that of SPF mice after intranasal instillation to lipopolysaccharide (LPS), a component of Gram-negative bacteria. Toll-like receptor 4 (TLR4) was more abundantly expressed in the lungs of GF mice than those of SPF mice at steady state, which could predispose the innate immunity of GF mice to strongly react to the environmental stimuli. Lung explants were stimulated with different TLR agonists or infected with the human airways pathogen, respiratory syncytial virus (RSV), resulting in greater inflammation under almost all conditions for the GF explants. Finally, alveolar macrophages (AM) from GF mice presented a higher innate immune response upon RSV infection than those of SPF mice. Overall, these data suggest that the presence of microbiota in SPF mice induced a process of innate immune tolerance in the lungs by a mechanism which remains to be elucidated. Our study represents a step forward to establishing the link between the microbiota and the immune reactivity of the lungs.

The POTENTIAL OF BUTTERFLY PEA FLOWER METHANOL EXTRACT AS AN ANTIOXIDANT BY IN SILICO

Abstract: Butterfly pea flower (Clitoria ternatea L.) is a flowering plant from the Fabecea family that can grow vines. Butterfly pea flower are known to have chemical components that can act as antioxidants. This study aims to predict the potential of active compounds from methanol extract of butterfly pea flower in inhibiting reactive oxygen species (ROS) based on bond affinity (∆G), the value of Root Mean Square Deviation (RMSD) and their interactions. The method used was a computational method with in silico technique. The software used was Autodock Vina with visualization using the Biovia Discovery Studio Visualizer 2020. The enzyme receptor was NADPH Oxidase (NOX) obtained from Protein Data Bank and the test ligands were a chemical compound from methanol extract of butterfly pea flower. The results of the in silico study showed that the NO had a innate ligand, namely the GTP ligand which has a ∆G value of -7.3 kcal/mol, an RMSD value of 3.1111 Å and the interaction with the receptor that involves the presence of hydrogen bonds. Based on the results of the analysis of 11 test ligands, the chemical component of caffeine was predicted to have the most potential in inhibiting ROS compounds with a value of ∆G -5.4, RMSD value of 1.328 Å and had the same amino acid residue in hydrogen bonding, namely ASP118, and GLY15. The test ligand had the ability to inhibit ROS compounds with a lower level of stability than the innate ligand.
 Keywords: antioxidant; butterfly pea flower; in silico; NADPH oxidase, ROS

Deletion of the deISGylating enzyme USP18 enhances tumour cell antigenicity and radiosensitivity

BackgroundInterferon (IFN) signalling pathways, a key element of the innate immune response, contribute to resistance to conventional chemotherapy, radiotherapy, and immunotherapy, and are often deregulated in cancer. The deubiquitylating enzyme USP18 is a major negative regulator of the IFN signalling cascade and is the predominant human protease that cleaves ISG15, a ubiquitin-like protein tightly regulated in the context of innate immunity, from its modified substrate proteins in vivo.MethodsIn this study, using advanced proteomic techniques, we have significantly expanded the USP18-dependent ISGylome and proteome in a chronic myeloid leukaemia (CML)-derived cell line. USP18-dependent effects were explored further in CML and colorectal carcinoma cellular models.ResultsNovel ISGylation targets were characterised that modulate the sensing of innate ligands, antigen presentation and secretion of cytokines. Consequently, CML USP18-deficient cells are more antigenic, driving increased activation of cytotoxic T lymphocytes (CTLs) and are more susceptible to irradiation.ConclusionsOur results provide strong evidence for USP18 in regulating antigenicity and radiosensitivity, highlighting its potential as a cancer target.

Abstract LB-080: USP18 modulates the ISGylome, immune signaling and sensitizes tumor cells to irradiation and CTL recognition

Abstract The innate immune response plays a critical role in modulating the efficacy of and mechanisms conferring resistance to immune checkpoint blockade (ICB) therapies in humans. A major negative regulator of the Interferon (IFN) stimulated gene (ISG) pathway, is the ubiquitin-specific protease 18 (USP18). USP18 is the predominant human deubiquitylating enzyme that processes Interferon Stimulated Gene 15 ISG15, a ubiquitin-like protein that covalently modifies protein substrates, a tightly regulated process in the context of innate immunity. In this study, using advanced mass spectrometry and chemical biology tools, we defined the USP18 Interactome and ISG15ylome in chronic myeloid leukemia (CML)-derived cells (HAP1) treated with Interferon alpha (IFNα). Novel ISG15ylation targets were characterized that reduce the sensing of innate ligands and secretion of cytokines. In addition, we show that USP18 deletion leads to enhanced ISG15ylation profiles. Furthermore, we demonstrate that CML USP18-/- cells are more antigenic, leading to increased activation of cytotoxic T lymphocytes (CTLs), and are more susceptible to irradiation. Our results reinforce the role of USP18 as a key “brake” for inflammatory signals in tumor cells. As USP18 expression is upregulated in lung, breast and colon cancers, USP18 pharmacological inhibition may reflect a target opportunity in cancer immunotherapy. Citation Format: Adan Pinto-Fernandez, Helene Greenwood, Mariolina Salio, Jianzhou Chen, Thomas Partridge, George Vere, Hannah C. Scott, Andreas Damianou, Persephone Borrow, Ruth Muschel, Vincenzo Cerundolo, Benedikt M. Kessler. USP18 modulates the ISGylome, immune signaling and sensitizes tumor cells to irradiation and CTL recognition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-080.

Кишечный эндотоксин: иммунитет - воспаление - старение, как звенья одной цепи

Самообновление популяции является базисным элементом развития вида. В основе этого процесса лежит старение. Оно является связующим звеном между такими равновеликими фундаментальными понятиями как жизнь и смерть. В основе старения лежит низкоинтенсивное (с периодами обострений) воспаление, которое длительное время протекает бессимптомно, т.е. без каких-либо клинических проявлений. Наиболее яркий тому пример болезни атеросклеротической природы, которые неуклонно прогрессируют с возрастом и являются наиболее частой причины смерти. Неизбежность старения генетически предопределена природой иммунной системы и факторами регуляции её активности, среди которых особое место занимают кишечный эндотоксин и стресс (определяющий объём поступления ЛПС в общий кровоток). Главная роль адаптивного иммунитета - «клеточный надзор», реализуемый астрономическим числом рецепторов, которые ставят «чёрную метку» как на чужеродные, так и собственные антигены. Расплатой за соблюдение чистоты клеточного пула является аутоиммунный процесс, который, по-видимому, является основой низкоинтенсивного воспаления. Интенсивность этого самоуничтожающего процесса (скорость старения) определяется активностью врождённого иммунитета. Последняя зависит от концентрации лигандов врождённого иммунитета в общем кровотоке и способности иммунной системы на них реагировать. Существенные отличия механизма взаимодействия TLR4 c ЛПС от других TLR со своими лигандами, позволяет квалифицировать кишечный эндотоксин как «экзогормон»: адаптации, старения и эволюции. Таким образом, старение, как фундаментальный процесс, генетически предопределён самой природой иммунной системы и реализуется при участии кишечного эндотоксина и стресса, которые одновременно являются и облигатными факторами гомеостаза. Скорость старения может быть замедлена при помощи известных (и пока неведомых) средств нормализации показателей системной эндотоксинемии, которая является облигатным фактором гомеостаза и общей патологии. Self-renewal of a population is a basic element of species development. This process is based on aging. Aging is a link between such equipollent fundamental notions as life and death. The basis of aging is low-intensity (with periods of exacerbations) inflammation, which is asymptomatic for a long time, i.e., does not show any clinical manifestations. The brightest example of this is atherosclerosis, which steadily progresses with age and becomes the most common cause of death. The inevitability of aging is genetically determined by the nature of the immune system and factors that regulate its activity. A special place among these factors belongs to intestinal endotoxin and stress, which determines the amount of LPS entering the systemic circulation. The main role of adaptive immunity is “cell surveillance” mediated by an astronomical number of receptors that put a “black mark” on both self and foreign antigens. The payment for maintaining purity of the cell pool is the autoimmune process, which apparently underlies low-intensity inflammation. The intensity of this self-destructive process (aging rate) is determined by the activity of innate immunity. The innate immunity depends on concentration of innate immunity ligands in the systemic circulation and the ability of the immune system to respond to them. Significant differences in the TLR4 and LPS interaction mechanism from other TLRs interactions with their ligands allows to qualify the intestinal endotoxin as an “exohormone” of adaptation, aging, and evolution. Thus, aging as a fundamental process is genetically predetermined by the very nature of the immune system and occurs with participation of intestinal endotoxin and stress, which are also mandatory factors of homeostasis. Aging may be slowed using known and so far unknown means for reversing systemic endotoxinaemia, a mandatory factor of homeostasis and general pathology.

Innate signaling within the central nervous system recruits protective neutrophils

There is great interest in understanding how the central nervous system (CNS) communicates with the immune system for recruitment of protective responses. Infiltrating phagocytic monocytes and granulocytes are implicated in neuroinflammation in multiple sclerosis and its animal model experimental autoimmune encephalomyelitis (EAE). To investigate how CNS endogenous signals can be harnessed to promote anti-inflammatory programs, we have used a particulate Toll-like receptor 9 and nucleotide-oligomerization domain 2 bispecific innate ligand (MIS416), to address whether its phagocytosis within the CNS recruits protective myeloid cells. We find that MIS416 injected intrathecally into the cerebrospinal fluid via the cisterna magna induced a local chemokine response that recruited blood-derived monocytes and neutrophils to the CNS. These cells phagocytosed MIS416. The increase in EAE severity normally seen from time of onset did not occur in mice receiving MIS416. This suppression of disease symptoms was dependent on expression of the type I interferon receptor (IFNAR). Transfer of intrathecal MIS416-induced neutrophils suppressed EAE in recipient mice, while monocytes did not transfer protection. MIS416-induced neutrophils showed increased IL-10 expression that was IFNAR1-driven. In contrast to intrathecal administration, intravenous administration of MIS416 led to monocyte but not neutrophil infiltration to the CNS. We thus identify a CNS-intrinsic and -specific phagocytosis-induced recruitment of anti-inflammatory neutrophils that contribute to CNS homeostasis and may have therapeutic potential.

Unmasking cancer cell character.

Two studies show that cancer cells contain sufficient quantities of immunostimulatory nucleic acids to trigger interferon signalling, increase antitumour immunity and overcome resistance to checkpoint blockade. Loss of the RNA-editing enzyme ADAR1 enables tumour cells to sense these innate ligands.

Unmasking cancer cell character

Two studies show that cancer cells contain sufficient quantities of immunostimulatory nucleic acids to trigger interferon signalling, increase antitumour immunity and overcome resistance to checkpoint blockade. Loss of the RNA-editing enzyme ADAR1 enables tumour cells to sense these innate ligands.

Loss of ADAR1 in tumours overcomes resistance to immune checkpoint blockade.

Most patients with cancer either do not respond to immune checkpoint blockade or develop resistance to it, often because of acquired mutations that impair antigen presentation. Here we show that loss of function of the RNA-editing enzyme ADAR1 in tumour cells profoundly sensitizes tumours to immunotherapy and overcomes resistance to checkpoint blockade. In the absence of ADAR1, A-to-I editing of interferon-inducible RNA species is reduced, leading to double-stranded RNA ligand sensing by PKR and MDA5; this results in growth inhibition and tumour inflammation, respectively. Loss of ADAR1 overcomes resistance to PD-1 checkpoint blockade caused by inactivation of antigen presentation by tumour cells. Thus, effective anti-tumour immunity is constrained by inhibitory checkpoints such as ADAR1 that limit the sensing of innate ligands. The induction of sufficient inflammation in tumours that are sensitized to interferon can bypass the therapeutic requirement for CD8+ T cell recognition of cancer cells and may provide a general strategy to overcome immunotherapy resistance.

Abstract P3-05-01: Immune and transcriptional signatures of dendritic dell (DC) vaccination combined with chemotherapy in locally advanced, triple-negative breast cancer (TNBC) patients

Abstract BACKGROUND: Women with TNBC who do not achieve a pathologic complete response (pCR) with preoperative (preop) chemotherapy have a high risk of recurrence and death from BC. Immunotherapy is an attractive strategy as human BCs can be immunogenic, and enhancing the immune effector function may augment the cytotoxic effects of standard therapies. CLINICAL TRIAL: Following IRB-approved informed consent, 10 pts with locally advanced TNBC received preop dose-dense doxorubicin/cyclophosphamide (AC) followed by paclitaxel and carboplatin (TCb) chemotherapy, combined with antigen-loaded (TNBC antigens: Cyclin B1, WT1, and control viral antigens: CEF) autologous monocyte-derived DC vaccinations administered intratumorally and subcutaneously. DCs were generated with GM-CSF and type I interferon, loaded with antigen in the form of long peptides and activated with innate ligands (LPS and Clo75) and CD40 ligand. Vaccines were given at 4 time points prior to definitive surgery, and 3 times post-surgery, pre- and post-radiation therapy (RT). Safety was the primary study endpoint, and pCR rate in breast and axilla was a secondary endpoint. Correlative studies included assessment of immune response via ELISpot and transcriptional profiling of blood samples collected over time. RESULTS: All pts received the 4 vaccines during preop chemotherapy, and 7/10 received all 7 vaccines. At the time of definitive surgery, 4 pts achieved a pCR, 3 pts had macroscopic residual disease in the breast and axillary lymph nodes, and 3 pts had residual cancer burden scores of 1. As of June 1, 2017, all pts have been in follow-up for at least 1 year s/p completion of all vaccines, and 7/10 patients have no evidence of disease. To assess immune signatures with IFN-γ-ELISpot, PBMCs from baseline (BL) and several time points during vaccine treatment were cultured with control peptides or with peptide libraries covering vaccine antigens. Using a linear mixed model to account for repeated and missing observations we found statistically significant (α = 0.05) increases in Cyclin B1, WT1, and CEF ELISpots in at least 1 time point post-DC vaccination and in follow-up. Compared to BL, Cyclin B1 and WT1 increased at 3 day pre-RT in 8/10 and 7/10 pts, respectively. To assess transcriptional signatures, a linear mixed model was utilized to determine statistically significant differences in fold-change over time compared to the BL and healthy controls. Modular analysis of differentially expressed transcripts at BL revealed downregulation of transcripts related to the monocyte lineage in 7/10 pts. Longitudinal analysis revealed profound transcriptional changes during AC with downregulation of lymphocyte modules and upregulation of innate and inflammation modules. While the latter ones have normalized during TCb and follow-up, T cell module remained substantially downregulated throughout treatment and follow-up. CONCLUSIONS: Combination of preop chemotherapy and intratumoral and subcutaneous autologous DC vaccination is safe in locally advanced TNBC pts and is linked with profound changes in immune transcription signatures and with expansion of antigen-specific immune responses that can be detected in IFN-γ ELISpot. Citation Format: Palucka AK, Roberts LK, Zurawski SM, Tarnowski J, Turner J, Wang X, Blankenship D, Smith JL, Levin MK, Finholt JP, Burkeholder SB, Timis R, Muniz LS, Dao T, Grant M, Banchereau J, Zurawski G, Pascual V, O'Shaughnessy JA. Immune and transcriptional signatures of dendritic dell (DC) vaccination combined with chemotherapy in locally advanced, triple-negative breast cancer (TNBC) patients [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-05-01.

Innate immunity in sarcoidosis pathobiology.

The immunopathogenesis of sarcoidosis is considered to involve contributions from both adaptive and innate immune responses. Although the identification of adaptive responses to candidate pathogenic antigens will elucidate mechanisms that regulate inflammation in sarcoidosis, innate mechanisms likely represent the 'missing link' to the initiation, maintenance, and resolution of noncaseating granulomatous inflammation, the hallmark feature of sarcoidosis. Furthermore, environments that expose patients to candidate pathogenic antigens for sarcoidosis also provide opportunities for engagement with innate ligands. Several studies have identified enhanced responsiveness via pattern recognition receptor pathways, potentiating the local induction of cytokines relevant to granulomatous inflammation, such as through stimulation of nucleotide-binding oligomerization domain-like receptor and Toll-like receptor pathways. These pathways contribute to the inherent properties of granulomas including, in some cases, persistent localization of pathogens and antigen. Serum amyloid A has been identified to be abundant in sarcoidosis tissues, and this promiscuous host protein can serve as an innate ligand to regulate experimental granulomatous inflammation. Nascent evidence supports a potential role for alternatively activated macrophages to direct histopathological outcomes in sarcoidosis. Innate pathways deserve further investigation as potential therapeutic targets for inhibiting granuloma formation in sarcoidosis.